Calcimimetic and Calcilytic Drugs: Feats, Flops, and Futures.

The actions of extracellular Ca(2+) in regulating parathyroid gland and kidney functions are mediated by the extracellular calcium receptor (CaR), a G protein-coupled receptor. The CaR is one of the essential molecules maintaining systemic Ca(2+) homeostasis and is a molecular target for drugs useful in treating bone and mineral disorders. Ligands that activate the CaR are termed calcimimetics and are classified as either agonists (type I) or positive allosteric modulators (type II); calcimimetics inhibit the secretion of parathyroid hormone (PTH). Cinacalcet is a type II calcimimetic that is used to treat secondary hyperparathyroidism in patients receiving dialysis and to treat hypercalcemia in some forms of primary hyperparathyroidism. The use of cinacalcet among patients with secondary hyperparathyroidism who are managed with dialysis effectively lowers circulating PTH levels, reduces serum phosphorus and FGF23 concentrations, improves bone histopathology, and may diminish skeletal fracture rates and the need for parathyroidectomy. A second generation type II calcimimetic (AMG 416) is currently under regulatory review. Calcilytics are CaR antagonists that stimulate the secretion of PTH. Several calcilytic compounds have been evaluated as orally active anabolic therapies for postmenopausal osteoporosis but clinical development of all of them has been abandoned because they lacked clinical efficacy. Calcilytics might be repurposed for new indications like autosomal dominant hypocalcemia or other disorders beyond those involving systemic Ca(2+) homeostasis.

Allosteric modulators of the extracellular calcium receptor.

The extracellular calcium receptor (CaR) is a Family C G protein-coupled receptor that controls systemic Ca2+ homeostasis, largely by regulating the secretion of parathyroid hormone (PTH). Ligands that activate the CaR have been termed calcimimetics and are classified as either Type I (agonists) or Type II (allosteric activators) and effectively inhibit the secretion of PTH. CaR antagonists have been termed calcilytics and all act allosterically to stimulate secretion of PTH. The calcimimetic cinacalcet has been approved for treating parathyroid cancer and secondary hyperparathyroidism in patients on renal replacement therapy. Cinacalcet was the first allosteric modulator of a G proteincoupled receptor to achieve regulatory approval. This review will focus on the technologies used to discover and develop allosterically acting calcimimetics and calcilytics as novel therapies for bone and mineral-related disorders.

Parathyroid hormone fragments inhibit active hormone and hypocalcemia-induced 1,25(OH)2D synthesis.

Carboxyl (C)-terminal fragments of parathyroid hormone (PTH) oppose the calcemic, phosphaturic, and bone-resorbing effects of active hormone. To study the action of these fragments on 1,25(OH)(2)D (1,25-dihydroxyvitamin D) synthesis, we infused parathyroidectomized rats with human or rat active 1-34 or 1-84 PTH at doses selected to produce similar calcemic responses. Human active PTH influenced neither phosphate nor 1,25(OH)(2)D concentrations. However, active 1-34 rat PTH decreased phosphate to the same level as vehicle-treated rats and increased 1,25(OH)(2)D to very high levels, whereas active 1-84 PTH decreased phosphate but maintained 1,25(OH)(2)D. As the latter effect could have been due to C-terminal fragment generation during its metabolic breakdown, we infused a mixture of rat C-terminal fragments alone or with rat 1-34. The C-terminal fragments decreased 1,25(OH)(2)D and prevented hypocalcemic-induced 1,25(OH)(2)D synthesis. When infused with active rat 1-34, they lowered the 1,25(OH)(2)D level to that seen with intact rat 1-84. The C-terminal fragments did not influence either basal or rat 1-34- or 1-84-induced CYP27B1 mRNA levels, suggesting that their inhibitory effects on 1,25(OH)(2)D synthesis appears to be post-transcriptional.

The parathyroid polyhormone hypothesis revisited.

The parathyroid polyhormone hypothesis holds that peptides derived from the metabolism of parathyroid hormone (PTH) (so-called C-terminal fragments) are themselves biologically active and that their effects are mediated by a novel 'C-terminal receptor.' The evidence supporting these assertions is extensive but remains inconclusive. This Commentary focuses on in vivo pharmacology studies that provide information relevant to understanding the physiological significance of C-terminal fragments. The more recent studies of this sort provide compelling evidence that the bioactivity of C-terminal fragments is likely to become physiologically relevant in settings of secondary hyperparathyroidism. In this condition, circulating levels of C-terminal fragments greatly exceed those of PTH. There is convincing evidence that the hypocalcemic effect of C-terminal fragments results from direct actions on the skeleton that inhibit bone resorption. On the other hand, there are few if any results of in vivo studies suggesting a role for C-terminal fragments in more physiological settings, at least when parameters associated with systemic calcium homeostasis are assessed.

Misconceptions about calcimimetics.

Calcimimetics are ligands that activate the calcium receptor. Some are small molecules and of these, the most extensively studied are phenylalkylamines like cinacalcet. This compound is a positive allosteric modulator that selectively targets the parathyroid calcium receptor to inhibit the secretion of parathyroid hormone. Cinacalcet is the first calcimimetic compound to attain regulatory approval for the treatment of hyperparathyroidism resulting from end-stage renal disease. The discovery of calcimimetics and the receptor they act on are considered with the intent of extracting lessons relevant to medical research and the discovery of new drugs.

Calcimimetic and calcilytic drugs: just for parathyroid cells?

The cell surface calcium receptor (Ca2+ receptor) is a particularly difficult receptor to study because its primary physiological ligand, Ca2+, affects numerous biological processes both within and outside of cells. Because of this, distinguishing effects of extracellular Ca2+ mediated by the Ca2+ receptor from those mediated by other mechanisms is challenging. Certain pharmacological approaches, however, when combined with appropriate experimental designs, can be used to more confidently identify cellular responses regulated by the Ca2+ receptor and select those that might be targeted therapeutically. The Ca2+ receptor on parathyroid cells, because it is the primary mechanism regulating secretion of parathyroid hormone (PTH), is one such target. Calcimimetic compounds, which active this Ca2+ receptor and lower circulating levels of PTH, have been developed for treating hyperparathyroidism. The converse pharmaceutical approach, involving calcilytic compounds that block parathyroid cell Ca2+ receptors and stimulate PTH secretion thereby providing an anabolic therapy for osteoporosis, still awaits clinical validation. Although Ca2+ receptors are expressed throughout the body and in many tissues that are not intimately involved in systemic Ca2+ homeostasis, their physiological and/or pathological significance remains speculative and their value as therapeutic targets is unknown.

The search for calcium receptor antagonists (calcilytics).

The Ca(2+) receptor on the surface of parathyroid cells is the primary molecular entity regulating secretion of parathyroid hormone (PTH). Because of this, it is a particularly appealing target for new drugs intended to increase or decrease circulating levels of PTH. Calcilytic compounds are Ca(2+) receptor antagonists which increase the secretion of PTH. The first reported calcilytic compound was NPS 2143, an orally active molecule which elicits rapid, 3- to 4-fold increases in circulating levels of PTH. These rapid changes in plasma PTH levels are sufficient to increase bone turnover in ovariectomized, osteopenic rats. When administered together with an antiresorptive agent (estradiol), NPS 2143 causes an increase in trabecular bone volume and bone mineral density in osteopenic rats. The magnitude of these changes are far in excess of those caused by estradiol alone and are comparable with those achieved by daily administration of PTH or a peptide analog. These anabolic effects of NPS 2143 on bone are not associated with hyperplasia of the parathyroid glands. Calcilytic compounds can increase endogenous levels of circulating PTH to an extent that stimulates new bone formation. Such compounds could replace the use of exogenous PTH or its peptide fragments in treating osteoporosis.

Pharmacological regulation of parathyroid hormone secretion.

Parathyroid hormone (PTH) is the key endocrine factor regulating systemic Ca(2+) homeostasis. Elevated levels of circulating PTH increase bone turnover and, depending on the duration of elevation, will result in net anabolic or catabolic effects on the skeleton. Secretion of PTH from the parathyroid glands is regulated by small changes in circulating levels of Ca(2+) which are detected by a Ca(2+) receptor on the surface of parathyroid cells. This G protein-coupled receptor is the primary molecular entity used by parathyroid cells to regulate secretion of PTH. As such, the Ca(2+) receptor is a unique molecular target for new drugs capable of increasing or decreasing circulating levels of PTH. Compounds which activate the Ca(2+) receptor are termed calcimimetics and they inhibit the secretion of PTH; a calcimimetic compound is in late stage clinical trials for the treatment of both primary and secondary hyperparathyroidism. Conversely, calcilytic compounds, which are Ca(2+) receptor antagonists, stimulate secretion of PTH; a calcilytic compound is in early clinical development for the treatment of osteoporosis.

Calcilytic compounds: potent and selective Ca2+ receptor antagonists that stimulate secretion of parathyroid hormone.

Despite the discovery of many ions and molecules that activate the Ca2+ receptor, there are no known ligands that block this receptor. Reported here are the pharmacodynamic properties of a small molecule, NPS 2143, which acts as an antagonist at the Ca2+ receptor. This compound blocked (IC50 of 43 nM) increases in cytoplasmic Ca2+ concentrations [Ca2+]i elicited by activating the Ca2+ receptor in HEK 293 cells expressing the human Ca2+ receptor. NPS 2143, even when tested at much higher concentrations (3 microM), did not affect the activity of a number of other G protein-coupled receptors, including those most structurally homologous to the Ca2+ receptor. NPS 2143 stimulated parathyroid hormone (PTH) secretion from bovine parathyroid cells (EC50 of 41 nM) over a range of extracellular Ca2+ concentrations and reversed the effects of the calcimimetic compound NPS R-467 on [Ca2+]i and on secretion of PTH. When infused intravenously in normal rats, NPS 2143 caused a rapid and large increase in plasma levels of PTH. Ca2+ receptor antagonists are termed calcilytics and NPS 2143 is the first substance (either atomic or molecular) shown to possess such activity. The pharmacodynamic properties of NPS 2143 together with the recently demonstrated effects of this compound on bone formation support the view that orally active calcilytic compounds might provide a novel anabolic therapy for osteoporosis.

Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats.

Parathyroid hormone (PTH) is an effective bone anabolic agent, but it must be administered parenterally. An orally active anabolic agent would provide a valuable alternative for treating osteoporosis. NPS 2143 is a novel, selective antagonist (a "calcilytic") of the parathyroid cell Ca(2+) receptor. Daily oral administration of NPS 2143 to osteopenic ovariectomized (OVX) rats caused a sustained increase in plasma PTH levels, provoking a dramatic increase in bone turnover but no net change in bone mineral density. Concurrent oral administration of NPS 2143 and subcutaneous infusion of 17beta-estradiol also resulted in increased bone turnover. However, the antiresorptive action of estrogen decreased the extent of bone resorption stimulated by the elevated PTH levels, leading to an increase in bone mass compared with OVX controls or to either treatment alone. Despite the sustained stimulation to the parathyroid gland, parathyroid cells did not undergo hyperplasia. These data demonstrate that an increase in endogenous PTH secretion, induced by antagonism of the parathyroid cell Ca(2+) receptor with a small molecule, leads to a dramatic increase in bone turnover, and they suggest a novel approach to the treatment of osteoporosis.

The calcimimetic R-467 potentiates insulin secretion in pancreatic beta cells by activation of a nonspecific cation channel.

The extracellular, G protein-linked Ca(2+)-sensing receptor (CaSR), first identified in the parathyroid gland, is expressed in several tissues and cells and can be activated by Ca(2+) and some other inorganic cations and organic polycations. Calcimimetics such as NPS (R)-N-(3-phenylpropyl)-alpha-methyl-3-methoxybenzylamine hydrochloride (R-467), a phenylalkylamine, are thought to activate CaSR by allosterically increasing the affinity of the receptor for Ca(2+). When tested for its effect on insulin release in C57BL/6 mice, R-467 had no effect under basal conditions but enhanced both phases of glucose-stimulated release. The betaHC9 cell also responded to R-467 and to the enantiomer S-467 with a stimulation of insulin release. In subsequent studies with the betaHC9 cell, it was found that the stimulatory effect was due to activation of a nonspecific cation channel, depolarization of the beta-cell, and increased Ca(2+) entry. No other stimulatory mechanism was uncovered. The depolarization of the cell induced by the calcimimetic could be due to a direct action on the channel or via the CaSR. However, it appeared not to be mediated by G(i), G(o), G(q/11), or G(s). The novel mode of action of the calcimimetic, combined with the glucose-dependence of the stimulation on islets, raises the possibility of a totally new class of drugs that will stimulate insulin secretion during hyperglycemia but which will not cause hypoglycemia.

Daily intermittent decreases in serum levels of parathyroid hormone have an anabolic-like action on the bones of uremic rats with low-turnover bone and osteomalacia.

The calcium receptor agonist (calcimimetic) compound NPS R-568 causes rapid decreases in circulating levels of parathyroid hormone (PTH) in rats and humans. We hypothesized that daily intermittent decreases in serum PTH levels may have different effects on bone than do chronically sustained decreases. To test this hypothesis, we compared two NPS R-568 dosing regimens in rats with chronic renal insufficiency induced by two intravenous injections of adriamycin. Fourteen weeks after the second adriamycin injection, creatinine clearance was reduced by 52%, PTH levels were elevated approximately 2.5-fold, and serum 25(OH)D3 and 1,25(OH)2D3 levels were reduced substantially. Treatment by daily per os gavage, which decreased PTH levels intermittently, or continuous subcutaneous infusion, which resulted in a sustained suppression of serum PTH levels, then began for 8 weeks. Despite the hyperparathyroidism, the adriamycin-injected rats developed a low-turnover bone lesion with osteomalacia (fourfold increase in osteoid volume in the proximal tibial metaphysis) and osteopenia (67% decrease in cancellous bone volume and an 18% reduction in bone mineral density at the distal femur). Daily administered (but not infused) NPS R-568 significantly increased cancellous bone volume solely by normalizing trabecular thickness, and increased femoral bone mineral density by 14%. These results indicate that daily intermittent, but not sustained, decreases in PTH levels have an "anabolic-like" effect on bones with a low-turnover lesion in this animal model of chronic renal insufficiency.

Calcimimetic NPS R-568 prevents parathyroid hyperplasia in rats with severe secondary hyperparathyroidism.

UNLABELLED: Calcimimetic NPS R-568 prevents parathyroid hyperplasia in rats with severe secondary hyperparathyroidism. BACKGROUND: Secondary hyperparathyroidism (secondary HPT) in chronic renal insufficiency (CRI) is characterized by multiglandular hyperplasia. METHODS: In this study, we investigated the effects of the calcimimetic NPS R-568 on the parathyroid gland in rats with CRI induced by ligation of the renal arteries and severe secondary HPT induced by dietary phosphorus loading. Six days after surgery, high-phosphorus diet feeding was started, and NPS R-568 was administered to the rats for 56 days either by daily gavage (30 or 100 micromol/kg) or by continuous subcutaneous infusion (20 micromol/kg. day). RESULTS: After 54 days, serum PTH levels in vehicle-treated CRI rats were 1019 vs. 104 pg/mL in sham-operated controls. Infusion of NPS R-568 maintained serum PTH at levels comparable with those of sham-operated controls, whereas daily gavage also prevented much of the increase in CRI controls and decreased PTH levels intermittently in a dose-dependent fashion. Parathyroid gland enlargement was caused predominantly by hyperplasia. Total cell number per kg body wt was 3.5-fold higher in vehicle-treated CRI rats than in sham-operated controls. Both infusion and high-dose gavage of NPS R-568 completely prevented the increase in parathyroid cell number. CONCLUSION: These results demonstrate that the calcimimetic compound NPS R-568 can prevent both the increase in serum PTH levels and parathyroid hyperplasia in rats with CRI and severe secondary HPT. Moreover, these changes occurred despite decreases in serum 1, 25(OH)2D3 and increases in serum phosphate, suggesting a dominant role for the calcium receptor in regulating parathyroid cell proliferation.

The calcium receptor and calcimimetics.

Parathyroid cells can sense small changes in plasma Ca2+ levels by virtue of a cell surface Ca2+ receptor. Calcimimetics are newly synthesized compounds that act as agonists or positive allosteric modulators at the Ca2+ receptor and can suppress parathyroid hormone secretion. The first-generation calcimimetic, NPS R-568, has undergone clinical trials in primary hyperparathyroidism and in hyperparathyroidism secondary to chronic renal insufficiency. The data accumulated so far demonstrate that calcimimetics have potential as therapeutic agents for hyperparathyroidism and related bone diseases such as osteitis fibrosa.

The calcimimetic NPS R-568 decreases plasma PTH in rats with mild and severe renal or dietary secondary hyperparathyroidism.

NPS R-568 is a Ca2+ receptor agonist ("calcimimetic") compound that reduces circulating parathyroid hormone (PTH) levels in rats and humans with mild secondary hyperparathyroidism (secondary HPT) resulting from chronic renal insufficiency (CRI). These studies extend those observations to show that NPS R-568 is equally effective in decreasing plasma PTH and Ca2+ levels in rats with mild or severe secondary HPT, resulting either from CRI or from dietary calcium deficiency. Male rats were 5/6 nephrectomized and fed either normal chow or a high-phosphorus diet; other normal rats were fed a low-calcium diet. When secondary HPT had developed, NPS R-568 was administered and blood samples were collected for up to 6 h. PTH levels decreased to a minimum level within 30 min in both CRI and calcium deficiency models of secondary HPT. PTH and Ca2+ levels remained significantly depressed for >3 h after dosing. The percentage decrease in PTH levels was unaffected by the severity of secondary HPT or the basal plasma Ca2+ or phosphate levels. In rats with severe secondary HPT, the minimum plasma PTH level after NPS R-568 was greater than the basal level in mild secondary HPT. Thus, NPS R-568 is equally effective in suppressing plasma PTH and Ca2+ levels in rats with mild or severe renal or nutritional secondary HPT.

NPS R-568: a type II calcimimetic compound that acts on parathyroid cell calcium receptor of rats to reduce plasma levels of parathyroid hormone and calcium.

Calcimimetics like N-(3-[2-chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine (NPS R-568) potentiate the effects of extracellular Ca(2+) on parathyroid Ca(2+) receptors and inhibit parathyroid hormone (PTH) secretion in vitro. When administered by gavage to normal rats in this study, NPS R-568 caused a rapid, dose-dependent (ED(50), 1.1 +/- 0.7 mg/kg) decrease in PTH levels that was paralleled by a subsequent decrease in plasma Ca(2+) (ED(50), 10.4 +/- 3.7 mg/kg). At higher doses (>/=3.3 mg/kg), PTH was reduced to a minimum level within 15 min, the duration of which was dose dependent. With doses of 10 to 100 mg/kg, the hypocalcemia was rapid in onset (<30 min) and, at 33 to 100 mg/kg, persisted for >24 h. Neither the magnitude nor the kinetics of the hypocalcemic response was affected by total nephrectomy, demonstrating that NPS R-568 does not induce hypocalcemia by acting on renal Ca(2+) receptors to increase Ca(2+) excretion. In contrast, parathyroidectomy (intact thyroid) abolished the hypocalcemic response to NPS R-568, regardless of whether the rats were hypocalcemic or rendered acutely normo- or hypercalcemic by calcium infusion before dosing. These data show that the parathyroid Ca(2+) receptor can be selectively activated in vivo with a small organic compound to decrease plasma levels of PTH and Ca(2+) and thus define the mechanism of action of this compound in vivo. Moreover, the data add pharmacological support to the view that the Ca(2+) receptor is the primary molecular entity regulating systemic Ca(2+) homeostasis.

Calcimimetic compound NPS R-568 stimulates calcitonin secretion but selectively targets parathyroid gland Ca(2+) receptor in rats.

N-(3-[2-Chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine (NPS R-568) is an orally active compound that activates Ca(2+) receptors on parathyroid cells and rapidly suppresses plasma levels of parathyroid hormone (PTH) and Ca(2+) (ED(50), 1 and 10 mg/kg, respectively). We now show that increased calcitonin secretion contributes to NPS R-568-induced hypocalcemia. In parathyroidectomized thyroid-intact rats in which normocalcemia was restored by PTH infusion, NPS R-568 rapidly reduced plasma Ca(2+) levels, indicating that decreased PTH secretion was not solely responsible for the hypocalcemia seen in normal animals. NPS R-568 decreased plasma Ca(2+) levels in thyroidectomized parathyroid-intact rats, but the rate of onset of hypocalcemia was slower than in controls. In contrast, NPS R-568 had no effect on plasma Ca(2+) levels in PTH-infused, thyroparathyroidectomized rats, providing evidence that increased calcitonin secretion caused the hypocalcemia in PTH-infused parathyroidectomized rats. NPS R-568 rapidly increased plasma calcitonin levels to a peak at 10 to 20 min after oral dosing (ED(50) 40 mg/kg). NPS R-568 did not affect the rate of disappearance of (45)Ca from blood, indicating that hypocalcemia resulted from decreased influx of Ca(2+) into the circulation and not from increased efflux. This suggests that NPS R-568-induced hypocalcemia resulted solely from reduced efflux of Ca(2+) from bone after increased calcitonin and reduced PTH secretion. Thus, NPS R-568 causes hypocalcemia by activating Ca(2+) receptors on C cells and parathyroid cells; however, NPS R-568 is about 40 times more potent in reducing PTH levels than in increasing calcitonin levels.

Domains determining ligand specificity for Ca2+ receptors.

The Ca2+ receptor is a G protein-coupled receptor that enables parathyroid cells and certain other cells in the body to respond to changes in the level of extracellular Ca2+. The Ca2+ receptor is a member of a family of G protein-coupled receptors that includes metabotropic glutamate receptors (mGluRs), gamma-aminobutyric acidB receptors, and putative pheromone receptors. As a family, these receptors are characterized by limited sequence homology and an unusually large putative extracellular domain (ECD). The ECD of the mGluRs is believed to determine agonist selectivity, but the functions of the structural domains of the Ca2+ receptor are not known. To identify structural determinants for cation recognition and activation of the Ca2+ receptor (and to further study the mGluRs), two chimeric receptors were constructed in which the large ECD of the Ca2+ receptor and the mGluR1 were interchanged. When expressed in Xenopus laevis oocytes, one of these chimeras, named CaR/mGluR1 [ECD of the Ca2+ receptor and transmembrane domain (TMD) of the mGluR1], responded to cation agonists (Gd3+, Ca2+, neomycin) of the Ca2+ receptor at concentrations similar to those necessary for activation of the native Ca2+ receptor. A reciprocal construct, named mGluR1/CaR (ECD of the mGluR1 and TMD of the Ca2+ receptor), was responsive to mGluR agonists but was much less sensitive to two of three cation agonists known to activate the Ca2+ receptor. A deletion construct of the Ca2+ receptor (DeltantCaR), which lacked virtually the entire ECD, was only activated by one of three agonists tested. These results suggest that the primary determinants for agonist activation of both the Ca2+ receptor and the mGluRs are found in the large ECD and that the Ca2+ receptor is possibly distinguished from the mGluRs in that it may contain sites in the TMD that permit activation by certain cation agonists.